P752: TARGETING S100A9 IN THE MYELODYSPLASTIC INFLAMMATORY BONE MARROW MICROENVIRONMENT BY TASQUINIMOD IMPROVES THE SUPPORTIVE FUNCTION OF MESENCHYMAL STROMAL CELLS
Authors: M. Wobus, E. Balaian, M. Lissner, R. Towers, U. Oelschlägel, R. Wehner, T. Chavakis, M. Törngren, H. Eriksson, E. Bondesson, U. Platzbecker, M. Bornhäuser, K. Sockel
Journal: HemaSphere (2022)

Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species (ROS). Tasquinimod (TASQ, Active Biotech) is a novel, small molecule inhibitor of S100A9 which has been investigated in several solid tumor entities and is currently under investigation in a phase Ib/IIa trial of patients with relapsed/refractory multiple myeloma. So far, little is known about its effects on myeloid malignancies. Aims: We aimed to investigate the role of S100A9 in cellular models of MDS and the in vitro potential of TASQ to target S100A9 within the MDS microenvironment. Methods: Mesenchymal stromal cells (MSCs) from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) ± TASQ (10µM). Subsequently, TLR4 downstream and proinflammatory signaling was analyzed by Western blot and real-time PCR. ROS levels were determined with a CellROX Flow Cytometry Kit (Thermo Fisher). Moreover, MSC differentiation and colony-forming unit-fibroblast (CFU-F) capacity were tested. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Results: Exposure of MDS and healthy MSCs to S100A9 induced TLR4 downstream signaling as demonstrated by increased expression of IRAK1 and NF-kB-p65 as well as gasdermin, an inductor of pyroptosis. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. In line with this, OXPHOS and ROS levels were decreased after addition of TASQ. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1β and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50% both at the mRNA and protein level. Moreover, addition of TASQ to MDS MSC cultures resulted in a significantly higher CFU-F number as well as abolished blocked the excessed adipogenic differentiation. In co-cultures with HSPCs, we observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs, which could be restored by TASQ pre-treatment. Summary/Conclusion: We provide evidence that TASQ mitigates the pathological inflammasome activation in the myelodysplastic BM niche in vitro by inhibition of the S100A9-mediated TLR4 signalling as well as its effects on NF-kB-p65 transcription and PD-L1 expression, resulting in improved hematopoietic support by MSCs, which suggests a beneficial effect in cytopenic MDS patients.